Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
  • A61K31/554—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having at least one nitrogen and one sulfur as ring hetero atoms, e.g. clothiapine, diltiazem
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D281/00—Heterocyclic compounds containing rings of more than six members having one nitrogen atom and one sulfur atom as the only ring hetero atoms
  • C07D281/02—Seven-membered rings
  • C07D281/04—Seven-membered rings having the hetero atoms in positions 1 and 4
  • C07D281/08—Seven-membered rings having the hetero atoms in positions 1 and 4 condensed with carbocyclic rings or ring systems
  • C07D281/10—Seven-membered rings having the hetero atoms in positions 1 and 4 condensed with carbocyclic rings or ring systems condensed with one six-membered ring
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D285/00—Heterocyclic compounds containing rings having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by groups C07D275/00 - C07D283/00
  • C07D285/36—Seven-membered rings

Definitions

• the invention relates to 1,5-benzothiazepine and 1,2,5-benzothiadiazepine derivatives of formula (I). These compounds are bile acid modulators having apical sodium-dependent bile acid transporter (ASBT) and/or liver bile acid transport (LBAT) inhibitory activity.
• ASBT apical sodium-dependent bile acid transporter
• LBAT liver bile acid transport
• the invention also relates to pharmaceutical compositions comprising these compounds and to these compounds for use in the treatment of cardiovascular diseases, fatty acid metabolism and glucose utilization disorders, gastrointestinal diseases and liver diseases.
• the 1,5-benzothiazepine and 1,2,5-benzothiadiazepine compounds of formula (I), or pharmaceutically acceptable salts thereof are inhibitors of the apical sodium-dependent bile acid transporter (ASBT inhibitors), of the liver bile acid transporter (LBAT inhibitors), or of both the apical sodium-dependent bile acid and liver bile acid transporters (dual ASBT/LBAT inhibitors). They are therefore useful in the treatment or prevention of conditions, disorders and diseases wherein inhibition of bile acid circulation is desirable, such as cardiovascular diseases, fatty acid metabolism and glucose utilization disorders, gastrointestinal diseases and liver diseases.
• ASBT inhibitors apical sodium-dependent bile acid transporter
• LBAT inhibitors liver bile acid transporter
• dual ASBT/LBAT inhibitors dual ASBT/LBAT inhibitors
• Cardiovascular diseases and disorders of fatty acid metabolism and glucose utilization include, but are not limited to, hypercholesterolemia; disorders of fatty acid metabolism; type 1 and type 2 diabetes mellitus; complications of diabetes, including cataracts, micro- and macrovascular diseases, retinopathy, neuropathy, nephropathy and delayed wound healing, tissue ischaemia, diabetic foot, arteriosclerosis, myocardial infarction, acute coronary syndrome, unstable angina pectoris, stable angina pectoris, stroke, peripheral arterial occlusive disease, cardiomyopathy, heart failure, heart rhythm disorders and vascular restenosis; diabetes-related diseases such as insulin resistance (impaired glucose homeostasis), hyperglycemia, hyperinsulinemia, elevated blood levels of fatty acids or glycerol, obesity, dyslipidemia, hyperlipidemia including hypertriglyceridemia, metabolic syndrome (syndrome X), atherosclerosis and hypertension; and for increasing high density lipoprotein levels.
• hypercholesterolemia disorders of fatty acid metabolism
• Gastrointestinal diseases and disorders include constipation (including chronic constipation, functional constipation, chronic idiopathic constipation (CIC), intermittent/sporadic constipation, constipation secondary to diabetes mellitus, constipation secondary to stroke, constipation secondary to chronic kidney disease, constipation secondary to multiple sclerosis, constipation secondary to Parkinson's disease, constipation secondary to systemic sclerosis, drug induced constipation, irritable bowel syndrome with constipation (IBS-C), irritable bowel syndrome mixed (IBS-M), pediatric functional constipation and opioid induced constipation); Crohn's disease; primary bile acid malabsorption; irritable bowel syndrome (IBS); inflammatory bowel disease (IBD); ileal inflammation; and reflux disease and complications thereof, such as Barrett's esophagus, bile reflux esophagitis and bile reflux gastritis.
• constipation including chronic constipation, functional constipation, chronic idiopathic constip
• a liver disease as defined herein is any disease in the liver and in organs connected therewith, such as the pancreas, portal vein, the liver parenchyma, the intrahepatic biliary tree, the extrahepatic biliary tree, and the gall bladder.
• a liver disease a bile acid-dependent liver disease.
• Liver diseases and disorders include, but are not limited to, an inherited metabolic disorder of the liver; inborn errors of bile acid synthesis; congenital bile duct anomalies; biliary atresia; post-Kasai biliary atresia; post-liver transplantation biliary atresia; neonatal hepatitis; neonatal cholestasis; hereditary forms of cholestasis; cerebrotendinous xanthomatosis; a secondary defect of BA synthesis; Zellweger's syndrome; cystic fibrosis-associated liver disease; alpha1-antitrypsin deficiency; Alagilles syndrome (ALGS); Byler syndrome; a primary defect of bile acid (BA) synthesis; progressive familial intrahepatic cholestasis (PFIC) including PFIC-1, PFIC-2, PFIC-3 and non-specified PFIC, post-biliary diversion PFIC and post-live
• Other diseases that may be treated or prevented by the compounds of formula (I), or pharmaceutically acceptable salts thereof, include hyperabsorption syndromes (including abetalipoproteinemia, familial hypobetalipoproteinemia (FHBL), chylomicron retention disease (CRD) and sitosterolemia); hypervitaminosis and osteopetrosis; hypertension; glomerular hyperfiltration; polycystic kidney disease (PKD), including autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD); and pruritus of renal failure.
• the compounds are also useful in the protection against liver- or metabolic disease-associated kidney injury.
• NTCP Na + -taurocholate cotransporting polypeptide
• ASBT apical sodium dependent bile acid transporter
• IBAT ileal bile acid transporter
• NTCP2 apical sodium dependent bile acid transporter
• bile acids are efficiently extracted from portal blood by the liver bile acid transporter (LBAT) and re-secreted across the canalicular membrane by the bile salt export pump (BSEP; gene symbol ABCB11 ).
• BSEP bile salt export pump
• the reabsorption of bile acids in the ileum is handled by the apical sodium-dependent bile acid transporter (ASBT), where it is commonly referred to as ileal bile acid transporter (IBAT).
• ASBT apical sodium-dependent bile acid transporter
• IBAT ileal bile acid transporter
• Both LBAT and ASBT function as electrogenic sodium-solute cotransporters that move two or more Na + ions per molecule of solute.
• Xenobiotics and endobiotics are taken up by the liver from portal blood and secreted into bile by distinct transport proteins with individualized substrate specificities.
• Glycine- and taurine-conjugated bile acids exist in anionic form and are unable to cross membranes by diffusion, and thus, are completely dependent on membrane transport proteins to enter or exit the hepatocyte ( Kosters and Karpen, Xenobiotica 2008, vol. 38, p. 1043-1071 ).
• ASBT and LBAT prefer glycine- and taurine-conjugated bile salts over their unconjugated counterparts and demonstrate a higher affinity for dihydroxy bile salts than for trihydroxy bile salts. No non-bile acid substrates have been identified for ASBT yet, however, LBAT was also found to transport a variety of steroid sulfates, hormones and xenobiotics.
• LBAT is not as thoroughly characterized as ASBT in terms of drug inhibition requirements.
• Dong et al. have identified FDA approved drugs that inhibit human LBAT and compared LBAT and ASBT inhibition requirements.
• the common feature pharmacophore indicated that two hydrophobes and one hydrogen bond acceptor were important for inhibition of LBAT. From 72 drugs screened in vitro, a total of 31 drugs inhibited LBAT, while 51 drugs (i.e. more than half) inhibited ASBT.
• ASBT unexpectedly was more permissive to drug inhibition than was LBAT, and this may be related to LBAT's possessing fewer pharmacophore features ( Dong et al., Mol. Pharm. 2013, vol. 10, p. 1008-1019 ).
• Vaz et al. describe the identification of LBAT deficiency as a new inborn error of metabolism with a relatively mild clinical phenotype.
• the identification of LBAT deficiency confirms that this transporter is the main import system for conjugated bile salts into the liver, but also indicates that auxiliary transporters are able to sustain the enterohepatic cycle in its absence ( Vaz et al., Hepatology 2015, vol. 61, p. 260-267 ).
• Liu et al. describe the identification of a new type of hypercholanemia that is associated with homozygosity for the p.Ser267Phe mutation in SLC10A1 (LBAT).
• the allele frequency of this mutation in gene SLC10A1 varies in different populations, with the highest incidence occurring in Southern China (8% and 12% in Chinese Han and Dai respectively) and in Vietnam (11%).
• This "hidden" hypercholanemia was believed to affect 0.64% of the Southern Han, 1.44% of the Dai Chinese population, and 1.21% of the Vietnamese population.
• An increase in conjugated and unconjugated serum BA levels in the homozygous individuals was also observed.
• LBAT has been found to be downregulated in several forms of cholestatic liver injury and cholestasis, whereas ASBT has been found to be downregulated in a variety of gastrointestinal disorders such as Crohn's disease, primary bile acid malabsorption, inflammatory bowel disease, and ileal inflammation but upregulated in cholestasis.
• LBAT also functions as a cellular receptor for viral entry of the hepatitis B virus (HBV) and hepatitis D virus (HDV), which in turn is the major cause of liver disease and hepatocellular carcinoma.
• HBV hepatitis B virus
• HDV hepatitis D virus
• ASBT inhibition has been investigated for decreasing plasma cholesterol levels and improving insulin resistance, as well as to relieving the hepatic bile acid burden in cholestatic liver disease.
• ASBT inhibition has been found to restore insulin levels and normoglycemia, thus establishing ASBT inhibition as a promising treatment for type 2 diabetes mellitus.
• ASBT inhibitors are also used for treatment of functional constipation.
• ASBT inhibitors need not be systemically available.
• ASBT is also expressed in the proximal tubule cells of the kidneys.
• ASBT inhibitors that are systemically available may therefore also inhibit the reuptake of bile acids in the kidneys. It is believed that this would lead to increased levels of bile acids in urine, and to an increased removal of bile acids from the body via the urine.
• Systemically available ASBT inhibitors that exert their effect not only in the ileum but also in the kidneys are therefore expected to lead to a greater reduction of bile acid levels than non-systemically available ASBT inhibitors that only exert their effect in the ileum.
• Compounds having a high ASBT inhibiting potency are particularly suitable for the treatment of liver diseases that cause cholestasis, such as progressive familial intrahepatic cholestasis (PFIC), Alagilles syndrome, biliary atresia and non-alcoholic steatohepatitis (NASH).
• PFIC progressive familial intrahepatic cholestasis
• Alagilles syndrome biliary atresia
• NASH non-alcoholic steatohepatitis
• Biliary atresia is a rare pediatric liver disease that involves a partial or total blockage (or even absence) of large bile ducts. This blockage or absence causes cholestasis that leads to the accumulation of bile acids that damages the liver. In some embodiments, the accumulation of bile acids occurs in the extrahepatic biliary tree. In some embodiments, the accumulation of bile acids occurs in the intrahepatic biliary tree.
• the current standard of care is the Kasai procedure, which is a surgery that removes the blocked bile ducts and directly connects a portion of the small intestine to the liver. There are currently no approved drug therapies for this disorder.
• a compound of formula (I), or a pharmaceutically acceptable salt thereof for use in the treatment of biliary atresia.
• the subject has undergone the Kasai procedure prior to administration of a compound of formula (I), or a pharmaceutically acceptable salt thereof.
• the subject is administered a compound of formula (I), or a pharmaceutically acceptable salt thereof, prior to undergoing the Kasai procedure.
• the treatment of biliary atresia decreases the level of serum bile acids in the subject.
• the level of serum bile acids is determined by, for example, an ELISA enzymatic assay or the assays for the measurement of total bile acids as described in Danese et al., PLoS One. 2017, vol. 12(6): e0179200 .
• the level of serum bile acids can decrease by, for example, 10% to 40%, 20% to 50%, 30% to 60%, 40% to 70%, 50% to 80%, or by more than 90% of the level of serum bile acids prior to administration of a compound of formula (I), or a pharmaceutically acceptable salt thereof.
• the treatment of biliary atresia includes treatment of pruritus.
• PFIC is a rare genetic disorder that is estimated to affect between one in every 50,000 to 100,000 children born worldwide and causes progressive, life-threatening liver disease.
• PFIC PFIC
• Current therapies include Partial External Biliary Diversion (PEBD) and liver transplantation, however, these options can carry substantial risk of post-surgical complications, as well as psychological and social issues.
• PEBD Partial External Biliary Diversion
• TJP2 gene, NR1H4 gene or Myo5b gene mutations have been proposed to be causes of PFIC.
• some subjects with PFIC do not have a mutation in any of the ATP8B1, ABCB11, ABCB4, TJP2, NR1H4 or Myo5b genes. In these cases, the cause of the condition is unknown.
• Exemplary mutations of the ATP8B1 gene or the resulting protein are listed in Tables 2 and 3, with numbering based on the human wild type ATP8B1 protein (e.g., SEQ ID NO: 1) or gene (e.g., SEQ ID NO: 2).
• Exemplary mutations of the ABCB11 gene or the resulting protein are listed in Tables 4 and 5, with numbering based on the human wild type ABCB11 protein (e.g., SEQ ID NO: 3) or gene (e.g., SEQ ID NO: 4).
• an amino acid position in a reference protein sequence that corresponds to a specific amino acid position in SEQ ID NO: 1 or 3 can be determined by aligning the reference protein sequence with SEQ ID NO: 1 or 3 (e.g., using a software program, such as ClustalW2). Changes to these residues (referred to herein as "mutations") may include single or multiple amino acid substitutions, insertions within or flanking the sequences, and deletions within or flanking the sequences.
• an nucleotide position in a reference gene sequence that corresponds to a specific nucleotide position in SEQ ID NO: 2 or 4 can be determined by aligning the reference gene sequence with SEQ ID NO: 2 or 4 (e.g., using a software program, such as ClustalW2). Changes to these residues (referred to herein as "mutations") may include single or multiple nucleotide substitutions, insertions within or flanking the sequences, and deletions within or flanking the sequences. See also Kooistra, et al., "KLIFS: A structural kinase-ligand interaction database," Nucleic Acids Res. 2016, vol. 44, no. D1, pp. D365-D371 .
• the mutation in ATP8B1 is selected from L127P, G308V, T456M, D554N, F529del, I661T, E665X, R930X, R952X, R1014X, and G1040R.
• the mutation in ABCB11 is selected from A167T, G238V, V284L, E297G, R470Q, R470X, D482G, R487H, A570T, N591S, A865V, G982R, R1153C, and R1268Q.
• methods for treating PFIC can include administering a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, to a subject having a mutation associated with PFIC (e.g., a ATP8B1, ABCB11, ABCB4, TJP2, NR1H4 or Myo5b mutation).
• a mutation associated with PFIC e.g., a ATP8B1, ABCB11, ABCB4, TJP2, NR1H4 or Myo5b mutation.
• the mutation is a ATP8B1 or ABCB11 mutation.
• NASH is a common and serious chronic liver disease that resembles alcoholic liver disease, but that occurs in people who drink little or no alcohol.
• fat accumulation in the liver known as nonalcoholic fatty liver disease (NAFLD) or steatosis
• NAFLD nonalcoholic fatty liver disease
• other factors such as high LDL cholesterol and insulin resistance induce chronic inflammation in the liver and may lead to progressive scarring of tissue, known as fibrosis, and cirrhosis, followed eventually by liver failure and death.
• Patients with NASH have been found to have significantly higher total serum bile acid concentrations than healthy subjects under fasting conditions (2.2- to 2.4-fold increase in NASH) and at all post-prandial time points (1.7- to 2.2-fold increase in NASH).
• NASH is defined as the presence of ⁇ 5% hepatic steatosis and inflammation with hepatocyte injury (e.g., ballooning), with or without any liver fibrosis. NASH is also commonly associated with hepatic inflammation and liver fibrosis, which can progress to cirrhosis, end-stage liver disease, and hepatocellular carcinoma. While liver fibrosis is not always present in NASH, the severity of the fibrosis, when present, can be linked to long-term outcomes.
• the NAS is determined non-invasively, for example, as described in U.S. Application Publication No. 2018/0140219 . In some embodiments, the NAS is determined for a sample from the subject prior to administration of a compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, the NAS is determined during the period of time or after the period of time of administration of a compound of formula (I), or a pharmaceutically acceptable salt thereof.
• a lower NAS score during the period of time or after the period of time of administration of a compound of formula (I), or a pharmaceutically acceptable salt thereof compared to prior to administration of the compound of formula (I), or a pharmaceutically acceptable salt thereof indicates treatment of NAFLD (e.g., NASH).
• NAFLD e.g., NASH
• a decrease in the NAS by 1, by 2, by 3, by 4, by 5, by 6, or by 7 indicates treatment of NAFLD (e.g., NASH).
• the NAS following administration of a compound of formula (I), or a pharmaceutically acceptable salt thereof is 7 or less.
• Additional approaches of assessing and evaluating NASH in a subject include determining one or more of hepatic steatosis (e.g., accumulation of fat in the liver); hepatic inflammation; biomarkers indicative of one or more of liver damage, hepatic inflammation, liver fibrosis, and/or liver cirrhosis (e.g., serum markers and panels).
• physiological indicators of NASH can include liver morphology, liver stiffness, and the size or weight of the subject's liver.
• methods to assess NASH include magnetic resonance imaging, either by spectroscopy or by proton density fat fraction (MRI-PDFF) to quantify steatosis, transient elastography (FIBROSCAN ® ), hepatic venous pressure gradient (HPVG), hepatic stiffness measurement with MRE for diagnosing significant liver fibrosis and/or cirrhosis, and assessing histological features of liver biopsy.
• magnetic resonance imaging is used to detect one or more of steatohepatitis (NASH-MRI), liver fibrosis (Fibro-MRI), and steatosis. See, for example, U.S. Application Publication Nos. 2016/146715 and 2005/0215882 .
• treatment of NASH comprises a decrease of one or more symptoms associated with NASH in the subject.
• Exemplary symptoms can include one or more of an enlarged liver, fatigue, pain in the upper right abdomen, abdominal swelling, enlarged blood vessels just beneath the skin's surface, enlarged breasts in men, enlarged spleen, red palms, jaundice, and pruritus.
• the subject is asymptomatic.
• the total body weight of the subject does not increase.
• the total body weight of the subject decreases.
• the body mass index (BMI) of the subject does not increase.
• the body mass index (BMI) of the subject decreases.
• the waist and hip (WTH) ratio of the subject does not increase.
• the waist and hip (WTH) ratio of the subject decreases.
• hepatic steatosis is determined by one or more methods selected from the group consisting of ultrasonography, computed tomography (CT), magnetic resonance imaging, magnetic resonance spectroscopy (MRS), magnetic resonance elastography (MRE), transient elastography (TE) (e.g., FIBROSCAN ® ), measurement of liver size or weight, or by liver biopsy (see, e.g., Di Lascio et al., Ultrasound Med Biol. 2018, vol. 44(8), p. 1585-1596 ; Lv et al., J Clin Transl Hepatol. 2018, vol. 6(2), p. 217-221 ; Reeder et al., J Magn Reson Imaging. 2011, vol.
• CT computed tomography
• MRS magnetic resonance spectroscopy
• MRE magnetic resonance elastography
• TE transient elastography
• FIBROSCAN ® transient elastography
• a subject with greater than about 5% to about 33% hepatic steatosis has stage 1 hepatic steatosis
• a subject with about 33% to about 66% hepatic steatosis has stage 2 hepatic steatosis
• a subject with greater than about 66% hepatic steatosis has stage 3 hepatic steatosis.
• a reduction in the amount of hepatic steatosis by about 1% to about 50%, about 25% to about 75%, or about 50% to about 100% indicates treatment of NASH.
• a reduction in the amount of hepatic steatosis by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% indicates treatment of NASH.
• a decrease in the severity of hepatic inflammation by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% indicates treatment of NASH.
• the severity (e.g., stage) of fibrosis is determined by one or more methods selected from the group consisting of transient elastography (e.g., FIBROSCAN ® ), a fibrosis-scoring system, biomarkers of hepatic fibrosis (e.g., non-invasive biomarkers), and hepatic venous pressure gradient (HVPG).
• transient elastography e.g., FIBROSCAN ®
• biomarkers of hepatic fibrosis e.g., non-invasive biomarkers
• HVPG hepatic venous pressure gradient
• fibrosis scoring systems include the NAFLD fibrosis scoring system (see, e.g., Angulo et al., Hepatology 2007, vol. 45(4), p. 846-54 ), the fibrosis scoring system in Brunt et al., Am. J. Gastroenterol.
• the severity of fibrosis is determined prior to administration of a compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, the severity of fibrosis is determined during the period of time or after the period of time of administration of a compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, a decrease in the severity of fibrosis during the period of time or after the period of time of administration of a compound of formula (I), or a pharmaceutically acceptable salt thereof, compared to prior to administration of the compound of formula (I), or a pharmaceutically acceptable salt thereof, indicates treatment of NASH.
• a decrease by 1, 2, 3, or 4 stages is a decrease in the severity of the fibrosis.
• a decrease in the stage e.g., from stage 4 to stage 3, from stage 4 to stage 2, from stage 4 to stage 1, from stage 4 to stage 0, from stage 3 to stage 2, from stage 3 to stage 1, from stage 3 to stage 0, from stage 2 to stage 1, from stage 2 to stage 0, or from stage 1 to stage 0 indicates treatment of NASH.
• the presence of NASH is determined by one or more biomarkers indicative of one or more of liver damage, inflammation, liver fibrosis, and/or liver cirrhosis or scoring systems thereof.
• the severity of NASH is determined by one or more biomarkers indicative of one or more of liver damage, inflammation, liver fibrosis, and/or liver cirrhosis or scoring systems thereof.
• the level of the biomarker can be determined by, for example, measuring, quantifying, and monitoring the expression level of the gene or mRNA encoding the biomarker and/or the peptide or protein of the biomarker.
• the presence of fibrosis is determined by one or more of the FIB-4 score, a panel of biomarkers consisting of ⁇ 2-macroglobulin, haptoglobin, apolipoprotein A1, bilirubin, gamma glutamyl transpeptidase (GGT) combined with a subject's age and gender to generate a measure of fibrosis and necroinflammatory activity in the liver (e.g., FIBROTEST ® , FIBROSURE ® ), a panel of biomarkers consisting of bilirubin, gamma-glutamyltransferase, hyaluronic acid, ⁇ 2-macroglobulin combined with the subject's age and sex (e.g., HEPASCORE ® ; see, e.g., Adams et al., Clin.
• HEPASCORE ® see, e.g., Adams et al., Clin.
• the level of aspartate aminotransferase does not increase. In some embodiments, the level of aspartate aminotransferase (AST) decreases. In some embodiments, the level of alanine aminotransferase (ALT) does not increase. In some embodiments, the level of alanine aminotransferase (ALT) decreases.
• the "level" of an enzyme refers to the concentration of the enzyme, e.g., within blood. For example, the level of AST or ALT can be expressed as Units/L.
• the severity of fibrosis is determined by one or more of the FIB-4 score, a panel of biomarkers consisting of ⁇ 2-macroglobulin, haptoglobin, apolipoprotein A1, bilirubin, gamma glutamyl transpeptidase (GGT) combined with a subject's age and gender to generate a measure of fibrosis and necroinflammatory activity in the liver (e.g., FIBROTEST ® , FIBROSURE ® ), a panel of biomarkers consisting of bilirubin, gamma-glutamyltransferase, hyaluronic acid, ⁇ 2-macroglobulin combined with the subject's age and sex (e.g., HEPASCORE ® ; see, e.g., Adams et al., Clin.
• HEPASCORE ® see, e.g., Adams et al., Clin.
• hepatic inflammation is determined by the level of liver inflammation biomarkers, e.g., pro-inflammatory cytokines.
• biomarkers indicative of liver inflammation include interleukin-(IL) 6, interleukin-(IL) 1 ⁇ , tumor necrosis factor (TNF)- ⁇ , transforming growth factor (TGF)- ⁇ , monocyte chemotactic protein (MCP)-1, C-reactive protein (CRP), PAI-1, and collagen isoforms such as Col1a1, Col1a2, and Col4a1 (see, e.g., Neuman, et al., Can. J. Gastroenterol. Hepatol. 2014, vol. 28(11), p. 607-618 and U.S.
• the level of one or more biomarkers indicative of one or more of liver damage, inflammation, liver fibrosis, and/or liver cirrhosis is determined for a sample from the subject prior to administration of a compound of formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, the level of one or more biomarkers indicative of one or more of liver damage, inflammation, liver fibrosis, and/or liver cirrhosis is determined during the period of time or after the period of time of administration of a compound of formula (I), or a pharmaceutically acceptable salt thereof.
• a decrease in the level of one or more biomarkers indicative of one or more of liver damage, inflammation, liver fibrosis, and/or liver cirrhosis during the period of time or after the period of time of administration of a compound of formula (I), or a pharmaceutically acceptable salt thereof, compared to prior to administration of the compound of formula (I), or a pharmaceutically acceptable salt thereof, indicates treatment of NASH.
• the decrease in the level of one or more biomarkers indicative of one or more of liver damage, inflammation, liver fibrosis, and/or liver cirrhosis following administration of the compound of formula (I), or a pharmaceutically acceptable salt thereof is by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%.
• the level of one or more biomarkers indicative of one or more of liver damage, inflammation, liver fibrosis, and/or liver cirrhosis during the period of time of administration of a compound of formula (I), or a pharmaceutically acceptable salt thereof is by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%.
• the level of one or more biomarkers indicative of one or more of liver damage, inflammation, liver fibrosis, and/or liver cirrhosis after the period of time of administration of a compound of formula (I), or a pharmaceutically acceptable salt thereof is by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%.
• the treatment of NASH decreases the level of serum bile acids in the subject.
• the level of serum bile acids is determined by, for example, an ELISA enzymatic assay or the assays for the measurement of total bile acids as described in Danese et al., PLoS One. 2017, vol. 12(6): e0179200 .
• the level of serum bile acids can decrease by, for example, 10% to 40%, 20% to 50%, 30% to 60%, 40% to 70%, 50% to 80%, or by more than 90% of the level of serum bile acids prior to administration of a compound of formula (I), or a pharmaceutically acceptable salt thereof.
• the NASH is NASH with attendant cholestasis.
• cholestasis the release of bile, including bile acids, from the liver is blocked.
• Bile acids can cause hepatocyte damage (see, e.g., Perez MJ, Briz O. World J. Gastroenterol. 2009, vol. 15(14), p. 1677-1689 ) likely leading to or increasing the progression of fibrosis (e.g., cirrhosis) and increasing the risk of hepatocellular carcinoma (see, e.g., Sorrentino P et al., Dig. Dis. Sci. 2005, vol. 50(6), p. 1130-1135 and Satapathy SK and Sanyal AJ. Semin.
• the treatment of NASH includes treatment of pruritus.
• the treatment of NASH with attendant cholestasis includes treatment of pruritus.
• a subject with NASH with attendant cholestasis has pruritus.
• biomarkers for NASH are provided in Table 7.
• a panel including ⁇ 2-macroglobulin, haptoglobin, apolipoprotein A1, bilirubin, gamma glutamyl transpeptidase (GGT) combined with a subject's age and gender to generate a measure of fibrosis and necroinflammatory activity in the liver e.g., FIBROTEST ® , FIBROSURE ®
• Some compounds of formula (I), or pharmaceutically acceptable salts thereof, may be excreted in urine.
• the fraction of the compound that is excreted in urine is greater than about 0.2%, such as greater than about 0.4%, such as greater than about 0.6%, such as greater than about 0.8%, such as greater than about 1.0%, such as greater than about 2%, such as greater than about 3%, such as greater than about 5%, such as greater than about 7.5%, such as greater than about 10%, such as greater than about 15%, such as greater than about 20%, such as greater than about 30%, or such as greater than about 50%.
• Some compounds of formula (I), or pharmaceutically acceptable salts thereof, may cause renal excretion of bile salts.
• the fraction of circulating bile acids that is excreted by the renal route is greater than about 1 %, such as greater than about 2%, such as greater than about 5%, such as greater than about 7%, such as greater than about 10%, such as greater than about 15%, such as greater than about 20%, or such as greater than about 25%.
• Some compounds of formula (I), or pharmaceutically acceptable salts thereof, may show improved or optimal permeability.
• the permeability may be measured in Caco2 cells, and values are given as Papp (apparent permeability) values in cm/s.
• the permeability is greater than at least about 0.1 x 10 -6 cm/s, such as greater than about 0.2 x 10 -6 cm/s, such as greater than about 0.4 x 10 -6 cm/s, such as greater than about 0.7 x 10 -6 cm/s, such as greater than about 1.0 x 10 -6 cm/s, such as greater than about 2 x 10 -6 cm/s, such as greater than about 3 x 10 -6 cm/s, such as greater than about 5 x 10 -6 cm/s, such as greater than about 7 x 10 -6 cm/s, such as greater than about 10 x 10 -6 cm/s, such as greater than about 15 x 10 -6 cm/s.
• the oral bioavailability is greater than about 5%, such as greater than about 7%, such as greater than about 10%, such as greater than about 15%, such as greater than about 20%, such as greater than about 30%, such as greater than about 40%, such as greater than about 50 %, such as greater than about 60 %, such as greater than about 70% or such as greater than about 80%.
• the oral bioavailability is between about 10 and about 90%, such as between about 20 and about 80%, such as between about 30 and about 70% or such as between about 40 and about 60%.
• Some compounds of formula (I), or pharmaceutically acceptable salts thereof, may be a substrate to relevant transporters in the kidney.
• Some compounds of formula (I), or pharmaceutically acceptable salts thereof, may give rise to concentrations of bile acids in the intestine, the liver and in serum that do not cause adverse gastrointestinal effects.
• Some compounds of formula (I), or pharmaceutically acceptable salts thereof, may decrease the concentration of bile acids in the liver without causing gastrointestinal disorders such as diarrhoea.
• treatment refers to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof, as described herein.
• treatment may be administered after one or more symptoms have developed.
• treatment may be administered in the absence of symptoms.
• treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence.
• a suitable pharmaceutically acceptable salt of a compound of the invention is, for example, a base-addition salt of a compound of the invention which is sufficiently acidic, such as an alkali metal salt (e.g., a sodium or potassium salt), an alkaline earth metal salt (e.g., a calcium or magnesium salt), an ammonium salt, or a salt with an organic base which affords a physiologically acceptable cation, for example a salt with methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris-(2-hydroxyethyl)amine.
• an alkali metal salt e.g., a sodium or potassium salt
• an alkaline earth metal salt e.g., a calcium or magnesium salt
• an ammonium salt e.g., sodium or potassium salt
• a salt with an organic base which affords a physiologically acceptable cation, for example a salt with methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris
• Some compounds of formula (I), or pharmaceutically acceptable salts thereof may have chiral centres and/or geometric isomeric centres (E- and Z-isomers). It is to be understood that the invention encompasses all such optical isomers, diastereoisomers and geometric isomers that possess ASBT and/or LBAT inhibitory activity. The invention also encompasses any and all tautomeric forms of compounds of formula (I), or pharmaceutically acceptable salts thereof, that possess ASBT and/or LBAT inhibitory activity. Certain compounds of formula (I), or pharmaceutically acceptable salts thereof, may exist in unsolvated as well as solvated forms, such as, for example, hydrated forms. It is to be understood that the invention encompasses all such solvated forms that possess ASBT and/or LBAT inhibitory activity.
• the invention in another aspect, relates to a pharmaceutical composition
• a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.
• the excipients may e.g. include fillers, binders, disintegrants, glidants and lubricants.
• pharmaceutical compositions may be prepared in a conventional manner using conventional excipients.
• suitable fillers include, but are not limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose (such as lactose monohydrate), sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, dry starch, hydrolyzed starches and pregelatinized starch.
• binders include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (such as sucrose, glucose, dextrose, lactose and sorbitol), polyethylene glycol, waxes, natural and synthetic gums (such as acacia gum and tragacanth gum), sodium alginate, cellulose derivatives (such as hydroxypropylmethylcellulose (or hypromellose), hydroxypropylcellulose and ethylcellulose) and synthetic polymers (such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid/polymethacrylic acid copolymers and polyvinylpyrrolidone (povidone)).
• sugars such as sucrose, glucose, dextrose, lactose and sorbitol
• polyethylene glycol such as acacia gum and tragacanth gum
• suitable disintegrants include, but are not limited to, dry starch, modified starch (such as (partially) pregelatinized starch, sodium starch glycolate and sodium carboxymethyl starch), alginic acid, cellulose derivatives (such as sodium carboxymethylcellulose, hydroxypropyl cellulose, and low substituted hydroxypropyl cellulose (L-HPC)) and cross-linked polymers (such as carmellose, croscarmellose sodium, carmellose calcium and cross-linked PVP (crospovidone)).
• modified starch such as (partially) pregelatinized starch, sodium starch glycolate and sodium carboxymethyl starch
• alginic acid such as sodium carboxymethylcellulose, hydroxypropyl cellulose, and low substituted hydroxypropyl cellulose (L-HPC)
• cross-linked polymers such as carmellose, croscarmellose sodium, carmellose calcium and cross-linked PVP (crospovidone)
• glidants and lubricants include, but are not limited to, talc, magnesium stearate, calcium stearate, stearic acid, glyceryl behenate, colloidal silica, aqueous silicon dioxide, synthetic magnesium silicate, fine granulated silicon oxide, starch, sodium lauryl sulfate, boric acid, magnesium oxide, waxes (such as carnauba wax), hydrogenated oil, polyethylene glycol, sodium benzoate, polyethylene glycol, and mineral oil.
• the pharmaceutical composition may be conventionally coated with one or more coating layers.
• Enteric coating layers or coating layers for delayed or targeted release of the compound of formula (I), or pharmaceutically acceptable salts thereof, are also contemplated.
• the coating layers may comprise one or more coating agents, and may optionally comprise plasticizers and/or pigments (or colorants).
• Example of suitable coating agents include, but are not limited to, cellulose-based polymers (such as ethylcellulose, hydroxypropylmethylcellulose (or hypromellose), hydroxypropylcellulose, cellulose acetate phthalate, cellulose acetate succinate, hydroxypropyl methylcellulose acetate succinate and hydroxypropyl methylcellulose phthalate), vinyl-based polymers (such as polyvinyl alcohol) and polymers based on acrylic acid and derivatives thereof (such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid/polymethacrylic acid copolymers).
• cellulose-based polymers such as ethylcellulose, hydroxypropylmethylcellulose (or hypromellose), hydroxypropylcellulose, cellulose acetate phthalate, cellulose acetate succinate, hydroxypropyl methylcellulose acetate
• plasticizers include, but are not limited to, triethyl citrate, glyceryl triacetate, tributyl citrate, diethyl phthalate, acetyl tributyl citrate, dibutyl phthalate, dibutyl sebacate and polyethylene glycol.
• suitable pigments include, but are not limited to, titanium dioxide, iron oxides (such as yellow, brown, red or black iron oxides) and barium sulfate.
• the pharmaceutical composition may be in a form that is suitable for oral administration, for parenteral injection (including intravenous, subcutaneous, intramuscular and intravascular injection), for topical administration of for rectal administration.
• parenteral injection including intravenous, subcutaneous, intramuscular and intravascular injection
• topical administration of for rectal administration is in a preferred embodiment, the pharmaceutical composition is in a form that is suitable for oral administration, such as a tablet or a capsule.
• the dosage required for the therapeutic or prophylactic treatment will depend on the route of administration, the severity of the disease, the age and weight of the patient and other factors normally considered by the attending physician, when determining the appropriate regimen and dosage level for a particular patient.
• the amount of the compound to be administered will vary for the patient being treated, and may vary from about 1 ⁇ g/kg of body weight to about 50 mg/kg of body weight per day.
• a unit dose form such as a tablet or capsule, will usually contain about 1 to about 250 mg of active ingredient, such as about 1 to about 100 mg, or such as about 1 to about 50 mg, or such as about 1 to about 20 mg, e.g. about 2.5 mg, or about 5 mg, or about 10 mg, or about 15 mg.
• the daily dose can be administered as a single dose or divided into one, two, three or more unit doses.
• An orally administered daily dose of a bile acid modulator is preferably within about 0.1 to about 250 mg, more preferably within about 1 to about 100 mg, such as within about 1 to about 5 mg, such as within about 1 to about 10 mg, such as within about 1 to about 15 mg, or such as within about 1 to about 20 mg.
• the invention relates to a compound of formula (I), or a pharmaceutically acceptable salt thereof, for use as a medicament.
• the invention relates to a compound of formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of any of the diseases recited herein.
• the compounds of formula (I), or pharmaceutically acceptable salts thereof are administered in combination with at least one other therapeutically active agent, such as with one, two, three or more other therapeutically active agents.
• the compound of formula (I), or a pharmaceutically acceptable salt thereof, and the at least one other therapeutically active agent may be administered simultaneously, sequentially or separately.
• Therapeutically active agents that are suitable for combination with the compounds of formula (I) include, but are not limited to, known active agents that are useful in the treatment of any of the aforementioned conditions, disorders and diseases.
• compounds of formula (I), or pharmaceutically acceptable salts thereof are administered in combination with another ASBT inhibitor.
• ASBT inhibitors are disclosed in WO 93/16055 , WO 94/18183 , WO 94/18184 , WO 96/05188 , WO 96/08484 , WO 96/16051 , WO 97/33882 , WO 98/03818 , WO 98/07449 , WO 98/40375 , WO 99/35135 , WO 99/64409 , WO 99/64410 , WO 00/47568 , WO 00/61568 , WO 00/38725 , WO 00/38726 , WO 00/38727 , WO 00/38728 , WO 00/38729 , WO 01/66533 , WO 01/68096 , WO 02/32428 , WO 02/50051 , WO 03/020710
• ASBT inhibitors include 1,1-dioxo-3,3-dibutyl-5-phenyl-7-methylthio-8-(N- ⁇ (R)-1'-phenyl-1'-[N'-(carboxymethyl)carbamoyl]methyl ⁇ carbamoylmethoxy)-2,3,4,5-tetrahydro-1,5-benzothiazepine (elobixibat) and 1,1-dioxo-3,3-dibutyl-5-phenyl-7-methylthio-8-(N- ⁇ (R)- ⁇ -[N-((S)-1-carboxypropyl) carbamoyl]-4-hydroxybenzyl ⁇ carbamoylmethoxy)-2,3,4,5-tetrahydro-1,2,5-benzothiadiazepine (odevixibat).
• compounds of formula (I), or pharmaceutically acceptable salts thereof are administered in combination with a bile acid binder (also referred to as a bile acid sequestrant, or a resin), such as colesevelam, cholestyramine or cholestipol.
• a bile acid binder also referred to as a bile acid sequestrant, or a resin
• colesevelam, cholestyramine or cholestipol a bile acid binder
• the bile acid binder is formulated for colon release. Examples of such formulations are disclosed in e.g. WO 2017/138877 , WO 2017/138878 , WO 2019/032026 and WO 2019/032027 .
• compounds of formula (I), or pharmaceutically acceptable salts thereof are administered in combination with an HMG CoA reductase inhibitor, such as fluvastatin, lovastatin, pravastatin, simvastatin, atorvastatin, pitavastatin cerivastatin, mevastatin, rosuvastatin, bervastatin or dalvastatin, or a pharmaceutically acceptable salt thereof.
• an HMG CoA reductase inhibitor such as fluvastatin, lovastatin, pravastatin, simvastatin, atorvastatin, pitavastatin cerivastatin, mevastatin, rosuvastatin, bervastatin or dalvastatin, or a pharmaceutically acceptable salt thereof.
• compounds of formula (I), or pharmaceutically acceptable salts thereof are administered in combination with a cholesterol absorption inhibitor such as ezetimibe, or a pharmaceutically acceptable salt thereof.
• compounds of formula (I), or pharmaceutically acceptable salts thereof are administered in combination with a PPAR alpha agonist, including fibrates such as clofibrate, bezafibrate, ciprofibrate, clinofribrate, clofibride, fenofibrate, gemfibrozil, ronifibrate and simfribrate, or a pharmaceutically acceptable salt thereof.
• fibrates such as clofibrate, bezafibrate, ciprofibrate, clinofribrate, clofibride, fenofibrate, gemfibrozil, ronifibrate and simfribrate, or a pharmaceutically acceptable salt thereof.
• compounds of formula (I), or pharmaceutically acceptable salts thereof are administered in combination with a PPAR gamma agonist, including thiazolidinediones such as pioglitazone, rosiglitazone and lobeglitazone, or a pharmaceutically acceptable salt thereof.
• a PPAR gamma agonist including thiazolidinediones such as pioglitazone, rosiglitazone and lobeglitazone, or a pharmaceutically acceptable salt thereof.
• compounds of formula (I), or pharmaceutically acceptable salts thereof are administered in combination with a dual PPAR alpha/gamma agonist, including glitazars such as saroglitazar, aleglitazar, muraglitazar or tesaglitazar, or a pharmaceutically acceptable salt thereof.
• glitazars such as saroglitazar, aleglitazar, muraglitazar or tesaglitazar, or a pharmaceutically acceptable salt thereof.
• compounds of formula (I), or pharmaceutically acceptable salts thereof are administered in combination with a dual PPAR alpha/delta agonist, such as elafibranor.
• compounds of formula (I), or pharmaceutically acceptable salts thereof are administered in combination with a pan PPAR agonist (i.e. a PPAR agonist that has activity across all subtypes: ⁇ , y and ⁇ ), such as IVA337.
• a pan PPAR agonist i.e. a PPAR agonist that has activity across all subtypes: ⁇ , y and ⁇
• compounds of formula (I), or pharmaceutically acceptable salts thereof are administered in combination with a farnesoid X receptor (FXR) modulators, including FXR agonists such as cafestol, chenodeoxycholic acid, 6 ⁇ -ethyl-chenodeoxycholic acid (obeticholic acid; INT-747), fexaramine, tropifexor, cilofexor and MET409.
• FXR farnesoid X receptor
• compounds of formula (I), or pharmaceutically acceptable salts thereof are administered in combination with a TGR5 receptor modulator, including TGR5 agonists such as 6 ⁇ -ethyl-23(S)-methylcholic acid (INT-777).
• compounds of formula (I), or pharmaceutically acceptable salts thereof are administered in combination with a dual FXR/TGR5 agonist such as INT-767.
• compounds of formula (I), or pharmaceutically acceptable salts thereof are administered in combination with ursodeoxycholic acid (UDCA).
• compounds of formula (I), or pharmaceutically acceptable salts thereof are administered in combination with nor-ursodeoxycholic acid (nor-UDCA).
• compounds of formula (I), or pharmaceutically acceptable salts thereof are administered in combination with an FGF19 modulator, such as NGM282.
• compounds of formula (I), or pharmaceutically acceptable salts thereof are administered in combination with an integrin inhibitor, such as PLN-74809 and PLN-1474.
• compounds of formula (I), or pharmaceutically acceptable salts thereof are administered in combination with a CCR2/CCR5 inhibitor, such as cenicriviroc.
• compounds of formula (I), or pharmaceutically acceptable salts thereof are administered in combination with a caspase protease inhibitor, such as emricasan.
• compounds of formula (I), or pharmaceutically acceptable salts thereof are administered in combination with a stearoyl-CoA desaturase (SCD) Inhibitor, such as aramchol (arachidyl amido cholanoic acid).
• SCD stearoyl-CoA desaturase
• compounds of formula (I), or pharmaceutically acceptable salts thereof are administered in combination with an apoptosis signal-regulating kinase 1 (ASK1) inhibitor, such as selonsertib.
• ASK1 apoptosis signal-regulating kinase 1
• compounds of formula (I), or pharmaceutically acceptable salts thereof are administered in combination with an LOXL2 inhibitor, such as silane.
• compounds of formula (I), or pharmaceutically acceptable salts thereof are administered in combination with an ACC inhibitor, such as GS-0976.
• compounds of formula (I), or pharmaceutically acceptable salts thereof are administered in combination with a thyroid hormone receptor- ⁇ agonist, such as MGL3196.
• Enantiomer 1 Yield: 42% (4.3 g, off-white solid).
• Enantiomer 2 Yield: 99% (0.4 g, yellow solid).
• Enantiomer 1 of the title compound was obtained following the procedure as described for intermediate 12 above, starting from 0.1 g of enantiomer 1 of Intermediate 9.
• Enantiomer 2 of the title compound was obtained following the same procedure, but starting from 0.1 g of enantiomer 2 of Intermediate 9.
• the absolute configuration of the two enantiomers is not known.
• Enantiomer 2 Yield: 66% (0.13 g, white solid).
• DBAD (0.12 g, 0.54 mmol) was then added at 0 °C and the reaction mixture was stirred for 16 hours at room temperature. After completion of the reaction (monitored by TLC), the reaction mixture was diluted with water (5 mL) and the aqueous layer was extracted with ethyl acetate (2 x 5 mL). The combined organic layer was washed with water (5 mL) and brine (5 mL) and dried over anhydrous Na 2 SO 4 . The organic part was filtered and concentrated under vacuum. The resulting crude material was purified by Isolera column chromatography (eluent: 22% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound. Yield: 81% (0.16 g, white solid).
• Tris[2-(2-methoxyethoxy)ethyl]amine (1.30 g, 4.00 mmol) was then added under nitrogen atmosphere and the resulting reaction mixture was heated for 40 h at 135 °C. After completion of the reaction (monitored by UPLC), the reaction mixture was filtered through celite and the celite pad was washed with EtOAc (100 mL). The filtrate was concentrated under vacuum and the resulting crude material was purified Isolera column chromatography (eluent: 15-20% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound. Yield: 64% (5.5 g, off-white solid).
• DBAD (0.04 g, 0.19 mmol) was then added at 0 °C and the reaction mixture was stirred for 16 hours at room temperature. After completion of the reaction (monitored by TLC), the reaction mixture was diluted with water (5 mL) and the aqueous layer was extracted with ethyl acetate (2 x 5 mL). The combined organic layer was washed with water (5 mL) and brine (5 mL) and dried over anhydrous Na 2 SO 4 . The organic part was filtered and concentrated under vacuum. The resulting crude material was purified by Isolera column chromatography (eluent: 20-22% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compound. Yield: 76% (50 mg, white solid).
• Diastereoisomer 1 Yield: 43% (0.13 g, off-white gum).
• Diastereoisomer 2 Yield: 41% (0.13 g, off-white gum).
• Diastereoisomers 1 and 2 of the title compound were prepared from diastereoisomer 1 (0.13 g) and diastereoisomer 2 (0.13 g) of Intermediate 51, respectively, following the same procedure as described for Intermediate 52. After work-up of the reaction mixtures, the crude materials were purified by Isolera column chromatography (eluent: 22% EtOAc/PE; silica gel: 230-400 mesh) to afford the title compounds. The absolute configuration of the two diastereomers is not known. Diastereoisomer 1: Yield: 69% (95 mg, pale yellow solid).
• Stereoisomers 1 and 2 of the title compound were prepared from stereoisomer 1 (0.25 g) and stereoisomer 2 (0.24 g) of Intermediate 56, respectively, following the same procedure as described for Intermediate 57.
• the absolute configuration of the two stereoisomers is not known.
• Stereoisomer 1 Yield: 54.9% (0.15 g, white solid).
• 1 H-NMR 400 MHz, CDCl 3 ): ⁇ 7.39 (s, 1H), 7.22-7.20 (m, 2H), 6.98 (s, 1H), 6.83-6.78 (m, 1H), 6.67-6.65 (m, 2H), 4.44-4.38 (m, 2H), 427-4.25 (m,1H), 4.09-4.04 (m, 2H), 3.87-3.80 (m, 5H), 3.61 (s, 3H), 3.23 (s, 1H), 2.57 (s, 3H), 2.33 (s, 3H), 1.75-1.70 (m, 1H), 1.58 (s, 3H), 1.50-1.28 (m, 3H).
• Enantiomer 2 of the title compound was obtained following the same procedure, starting from 0.13 g of enantiomer 2 of Intermediate 13. The absolute configuration of the two enantiomers is not known.
• Enantiomer 1 Yield: 37% (20 mg, off-white solid).
• Enantiomer 2 Yield: 23% (15 mg, off-white solid).
• Enantiomer 1 Yield: 24% (15 mg, off-white solid).
• Enantiomer 2 Yield: 23% (15 mg, off-white solid).
• Enantiomer 1 Yield: 25% (10 mg, white solid).
• Enantiomer 1 Yield: 12% (5 mg, off-white solid).
• Diastereoisomers 1 and 2 of the title compound were prepared from diastereoisomer 1 (95 mg) and diastereoisomer 2 (90 mg) of Intermediate 54, respectively, following the same procedure as described for Example 21. After work-up of the reaction mixtures, the crude materials were purified by Isolera column chromatography (eluent: 4% MeOH/DCM; silica gel: 230-400 mesh) to afford the title compounds. The absolute configuration of the two diastereomers is not known.
• Diastereoisomer 1 Yield: 57% (55 mg, white solid).
• a cryopreserved vial of differentiated HepaRG cells (Biopredic International HPR116080) is thawed in HepaRG Thawing/Plating/General Purpose Medium (Biopredic International ADD670C) supplemented with 200 mM Glutamax (Gibco 35050061) following the protocol provided by Biopredic International.
• 70,000 cells per well are seeded in 96-wells plate (Corning CLS3809) in 100 ⁇ L of HepaRG Thawing/Plating/General Purpose Medium supplemented with 200 mM Glutamax and incubated at 37 °C in 5% CO 2 for 24 hours.
• the seeding media is replaced by HepaRG Maintenance/Metabolism Medium (Biopredic International ADD620C) and incubated for 6 days, with fresh HepaRG Maintenance/Metabolism Medium replenishment every 48 hours.
• HepaRG Maintenance/Metabolism Medium Biopredic International ADD620C
• incubation media is decanted from the wells and cells are washed two times with 250 ⁇ L of William's E Basal Media (Gibco 12551032). After decanting William's E Basal Media each time, plates are tapped against paper towel to ensure maximum removal of residual media.
• Incubation mix is prepared by adding test inhibitor dilutions (3-fold serial dilution in DMSO (Sigma D2650)) in William's E media (basal) containing 0.3 ⁇ M 3H-taurocholic acid (ARC ART-1368) and 7.5 ⁇ M cold taurocholic acid (Sigma T4009) (maintaining 0.2% final DMSO concentration). 50 ⁇ l of incubation mix containing test inhibitors is then added to the wells (in duplicate) and the plates are incubated for 30 minutes in 5% CO 2 incubator at 37 °C. After incubation, the reaction is stopped by keeping the plates on ice water mix for 2-3 minutes and the incubation mix is then aspirated completely from the wells.
• MicroScint-20 (PerkinElmer 6013621) is added to the wells and kept overnight at room temperature before reading the plates in TopCount NXT TM Microplate Scintillation and Luminescence Counter from PerkinElmer under 3H Test protocol (set at 120 seconds reading time per well, with normal plate orientation).
• mice Male mice (C57BL/6 or CD1) or Wistar rats of 8-9 weeks old were used.
• One group was administered a single intravenous dose of 1 mg/kg (vehicle 100% DMSO) through the tail vein and the other group was administered a single oral dose of 10 mg/kg through gavage needle.
• the group that was administered an oral dose was fasted overnight.
• Blood samples were collected after 0.083, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours following intravenous administration, and after 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours following oral administration. Blood samples were taken from saphenous vein. 0.2% EDTA was used as the anticoagulant.
• the samples were analyzed by a discovery grade bioanalytical method developed for the estimation of test compound in plasma, using an LC-MS/MS system.
• PD model Evaluation of test compound on total bile acids levels in male C57BL6 mice.
• mice of 8-9 weeks old are used to study the effect of bile acid modulators on bile acid levels.
• animals are randomized based on bodyweight into x experimental groups: (i) vehicle control, and (ii) test compound y mg/kg po once daily. Animals are treated with test compound for 7 days. On day 6 of the study, animals are transferred to a metabolic cage. On day 7, feces and urine are collected from each metabolic cage, followed by blood withdrawal from each animal through retro-orbital route. Animals are euthanized to collect kidney from each animal for further analysis. Bodyweight is measured twice weekly. Total bile acids in serum is measured in serum samples of day 7.
• Fecal bile acid excretion is measured in the fecal sample of day 7.
• Urine excretion of bile acids is measured in the sample of day 7.
• Kidney expression of ASBT, OSTa, OSTAb and MRP2 is quantified in the samples of day 7.

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